A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Abstract

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

“…Molecular modeling (11)(12)(13), FRET measurements (14)(15)(16), and biochemical approaches (5,17,18) are consistent with a model in which dimers or higher-order oligomers of rhodopsin, or class A GPCRs in general, constitute the functional unit for G protein activation (19)(20)(21). Although this paradigm has been questioned (10,22,23), a supramolecular organization of GPCRs indeed may enhance the performance and fidelity with which signals are transferred to the G protein in signal transduction modules (11,24). Using a simplified system, namely monomeric rhodopsin in detergent solution, we have tested whether such organization of rhodopsin is necessary for efficient catalytic activation of G t , i.e., nucleotide exchange in the ␣-subunit of G t by interaction with light-activated rhodopsin (R*).…”

“…However, other GPCRs also were found to associate as dimers and higher-order oligomers in living cells (see, e.g., refs. 6-8), although monomers prevail at low expression levels (9,10). Molecular modeling (11)(12)(13), FRET measurements (14)(15)(16), and biochemical approaches (5,17,18) are consistent with a model in which dimers or higher-order oligomers of rhodopsin, or class A GPCRs in general, constitute the functional unit for G protein activation (19)(20)(21).…”

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

“…Quantification, at any rate, is a hard task even in conventional FRET and BRET assays, particularly when they are based on genetically encoded labels [3], and the quantitative interpretation of the data is still highly controversial matter [48,49]. Even if only capable of qualitative information, this new strategy should allow building assays with unrivaled characteristics in terms of signal to noise ratio, accuracy and sensitivity.…”

Single-molecule analysis of fluorescently labeled G-protein–coupled receptors reveals complexes with distinct dynamics and organization

“…For family-A GPCRs, the situation is more controversial, even for well-studied members such as the β 1 -adrenergic receptor (β 1 AR) and the β 2 -adrenergic receptor (β 2 AR) (6-10). For instance, β 2 ARs have been suggested to be monomers (13), constitutive dimers (14,15), or higher-order oligomers (16). Similarly, β 1 ARs have been suggested to form either stable (15) or transient interactions (16).…”

“…However, even though these methods have provided important insights on GPCR di-/oligomerization, they are based on average proximity measurements and usually require high receptor expression levels. Moreover, an intense debate arose a few years ago on the possible occurrence of RET due to random collisions (13,22,23).…”